Truncated Latrunculins as Actin Inhibitors Targeting Plasmodium falciparum Motility and Host Cell Invasion

Article abstract of DOI:10.1021/acs.jmedchem.6b01109

Polymerization of the cytosolic protein actin is critical to cell movement and host cell invasion by the malaria parasite, Plasmodium falciparum. Any disruption to actin polymerization dynamics will render the parasite incapable of invading a host cell and thereby unable to cause infection. Here, we explore the potential of using truncated latrunculins as potential chemotherapeutics for the treatment of malaria. Exploration of the binding interactions of the natural actin inhibitor latrunculins with actin revealed how a truncated core of the inhibitor could retain its key interaction features with actin. This truncated core was synthesized and subjected to preliminary structure-activity relationship studies to generate a focused set of analogues. Biochemical analyses of these analogues demonstrate their 6-fold increased activity compared with that of latrunculin B against P. falciparum and a 16-fold improved selectivity ex vivo. These data establish the latrunculin core as a potential focus for future structure-based drug design of chemotherapeutics against malaria.

Full text of DOI:10.1021/acs.jmedchem.6b01109

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Article
Truncated Latrunculins as Actin Inhibitors Targeting
Plasmodium falciparum Motility and Host-Cell Invasion
Swapna Johnson, Raphaël Rahmani, Damien R. Drew, Melanie J. Williams,
Johnny X. Huang, Mark Dale Wilkinson, Yan Hong Tan, Christopher J
Tonkin, James G. Beeson, Jake Baum, Brian J. Smith, and Jonathan B. Baell
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01109 • Publication Date (Web): 09 Nov 2016
Downloaded from http://pubs.acs.org on November 10, 2016
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Truncated Latrunculins as Actin Inhibitors
Targeting Plasmodium falciparum Motility and
HostꢀCell Invasion
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†
,‡
‡
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¶
Swapna Johnson, Raphaël Rahmani, Damien R. Drew, Melanie J. Williams, Mark
#
¶,#
¥
¶
§
Wilkinson, Yan Hong Tan, Johnny X. Huang, Christopher J. Tonkin, James G. Beeson,
¶,# ,*
†,*
‡,*
Jake Baum, Brian J. Smith and Jonathan B. Baell
†
La Trobe Institute for Molecular Science, La Trobe University, Melbourne Victoria 3086,
Australia
‡
Monash Institute of Pharmaceutical Sciences, Monash University, Parkville Victoria 3052,
Australia
§
Burnet Institute, 85 Commercial Rd, Melbourne Victoria 3004, Australia. Central Clinical
School and Department of Microbiology, Monash University, Melbourne Victoria 3004,
Australia
¶
Walter and Eliza Hall Institute, 1G Royal Parade, Parkville Victoria 3052, Australia
#
Department of Life Sciences, Imperial College, London, South Kensington, SW7 2AZ, UK
¥
Institute for Molecular Bioscience, The University of Queensland, St Lucia Queensland
4
072, Australia
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KEYWORDS: Malaria, actin, latrunculin.
ABSTRACT: Polymerization of the cytosolic protein actin is critical to cell movement and
hostꢀcell invasion by the malaria parasite, Plasmodium falciparum. Any disruption to actin
polymerization dynamics will render the parasite incapable of invading a host cell and
thereby unable to cause infection. Here, we explore the potential of using truncated
latrunculins as potential chemotherapeutics for the treatment of malaria. Exploration of the
binding interactions of the natural actin inhibitor latrunculins, with actin revealed how a
truncated core of the inhibitor could retain its key interaction features with actin. This
truncated core was synthesised and subjected to preliminary structureꢀactivity relationship
studies to generate a focused set of analogues. Biochemical analyses of these analogues
demonstrate their 6ꢀfold increased activity compared with latrunculin B against Plasmodium
falciparum and a 16ꢀfold improved selectivity ex vivo. These data establish the latrunculin
core as a potential focus for future structureꢀbased drug design of chemotherapeutics against
malaria.
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INTRODUCTION
With many millions of new cases of malaria reported every year, the global burden of this
disease remains very high. The World Health Organization (WHO) reported 214 million
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cases of malaria in 2015 with approximately half a million deaths. Malaria disease is
primarily caused by parasites from the genus Plasmodium with Plasmodium falciparum
responsible for the vast majority of deaths. This single celled eukaryotic parasite is
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ꢀ5
transmitted by female Anopheles mosquitoes during an infectious blood feed. Although a
number of drugs are available that can effectively treat malaria, the growing resistance of the
parasites to these existing treatment regimes is a major issue for future efforts to achieve
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sustained control or reduction of the disease globally. The development of resistance implies
that the current drug targets are increasingly being compromised and hence there is an urgent
need to explore novel chemotherapeutic targets.
Actin is one of the most abundant proteins found in all eukaryotic cells, and is responsible
for a large number of cellular functions such as cell movement, chromosomal condensation,
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cytoplasmic streaming, cytokinesis and maintenance of the cytoskeletal structure. Actin can
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reversibly polymerise between its monomeric (Gꢀactin) and filamentous (Fꢀactin) form.
Importantly, actin dynamics is an essential element involved in the cell motility and host cell
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invasion of malarial parasites.
Malaria disease occurs during the bloodꢀstage of infection,
when the merozoite form of parasites invade red blood cells and replicate inside them, and
actin is crucial in this invasion process. As such, any disruption in the actin dynamics could
result in arrest of parasite movement, preventing it from causing disease.
A large number of natural products are known to bind to actins and thereby alter their
ability to polymerize or block their ATPase activity; these compounds are therefore
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cytotoxic. Such molecules have a specific target site on the actin molecule, often disrupting
the ability of Gꢀactin to form filaments, destabilizing the growing filament or stabilising the
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filament and subsequently preventing disassembly. Structural analysis of the interaction
between actin and bound cytotoxins reveals that several cytotoxin binding sites defined in
mammalian actin are located in areas where there is marked sequence difference between
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human and P. falciparum actin. Thus, compounds that selectively target such sites, with a
preference for residues characterising the P. falciparum actin over human actin, could be
used to selectively target parasite actinꢀdependent processes, arresting cell motility and
invasion and, as such, present a potentially potent therapeutic approach to stopping malaria
disease.
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Latrunculins (Figure 1) are macrolides initially isolated from the marine sponge
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Negombata magnifica found in the red sea
and are known to bind to the Gꢀactin
monomer in a 1:1 complex blocking the ability of actin to polymerize into the growing
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filament.
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Figure 1. Structures of latrunculin A (1) and B (2), the latrunculin core (3a) and the core
analogue substructure (3b).
The latrunculins specifically bind near the ATP binding cleft between subdomains D2 and
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D4 of the actin monomer and thereby sequester actin away from the free cytosolic pool in a
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fashion similar to that of monomer sequestering proteins. We set out to identify the key
differences in the latrunculinꢀ bound binding pocket of the human and the P. falciparum actin
in order to design a parasiteꢀspecific latrunculinꢀlike compound. Here we describe the first
steps in the development of the latrunculin actin binding interaction as a drug target for the
treatment of malaria. Specifically, we describe the discovery of truncated latrunculin
analogues based on a computer generated model, that when subjected to preliminary
Structure Activity Relationship (SAR) studies provide the framework for the synthesis of a
focused set of analogues with a 6ꢀfold increase in their general activity and a 16ꢀfold increase
in selectivity against P. falciparum parasites. Critically, we can provide a structural
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explanation for the SAR and selectivity of these truncated analogues based on computational
modelling using the parasite and human actin model structures.
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RESULTS AND DISCUSSION
Models of latrunculinꢀbound P. falciparum (Pf) and human (hs) actin were created using
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the ‘MODELLER’ program with the crystal structure of rabbit muscle actin with latrunculin
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B bound (PDB ID: 2Q0U ) as the template. The alignment of Pf and hs actin sequences with
that of rabbit muscle was guided by predictions of the secondary structure using the
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PSIPRED program, and by automated sequence alignment using ClustalW; the three
sequences share 79% identity and 92% similarity (Supplementary Information Figure S1).
The model of Pf actin is almost identical to the recently solved Xꢀray crystal structure of this
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protein without ligand bound (PDB ID: 4CBU ); the root mean square deviation from the
superimposition of Cα atoms is 0.9 Å, the most significant difference being displacement of
helix α1 required to expose the ligand binding cleft.
Comparison of the binding sites of the human and the P. falciparum actin and
identification of the key pharmacophores. Comparison of the homology models of the
human and P. falciparum actins revealed that latrunculin B lies close to the ATP binding site
and that the residues in the binding pocket are highly conserved. Only two residue differences
were identified within 7 Å distance of latrunculin B, the first being the replacement of
Arg206 in human actin with a Lys207 in P. falciparum actin, and a second difference where
Met16 in human actin is replaced by Asn17 in P. falciparum actin. While the ester carbonyl
of latrunculin B cannot form any hydrogen bonds with Met16 in the human actin, it can
engage in hydrogen bonding with Asn17 in the P. falciparum actin (Figure 2).
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Figure 2. Model of latrunculin B bound to P. falciparum actin. Latrunculin B is presented in
green (and heteroatom colors), while the interacting protein residue side chains are presented
in tan. Hydrogen bonds between latrunculin B and the protein are indicated by dashed lines.
Other interactions observed in the P. falciparum actin model include hydrogen bonds
between Glu208 and the hemiketal hydroxyl group, between Thr187 and the carbonyl of the
thiazolidinone, between Tyr70 and the oxygen atom of the tetrahydropyran (THP) ring, and
between Asp158 and the nitrogen atom of the thiazolidinone ring. These interactions are
conserved in the human actin as well. In both cases the macrocycle is involved in
hydrophobic interactions with the hydrophobic residues Pro33 and Ile35.
From these models it is clear that the thiazolidinone and the THP moiety together engage in
4 hydrogen bonds; this suggests that even in the absence of the macrocycle, a truncated
molecule could interact favourably with the actin protein. The retention of biological activity
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by smaller truncated fragments of a natural product is not unprecedented, with several
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examples apparent in the literature.
Previous studies have identified the thiazolidinone
and the THP ring as the key pharmacophores involved in mammalian actin binding, while the
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macrocyclic ring acts to stabilise the binding by forming hydrophobic interactions. Together
these insights suggest a truncated latrunculin core (Figure 1) that can be derivatised at the
secondary alcohol to mimic the macrocycle could retain latrunculinꢀlike activity;
significantly, removing the two Zꢀisomer double bonds of the macrocycle reduces synthetic
complexity.
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In consideration of the synthesis of the latrunculin core a number of important design
features were addressed. The latrunculin core possesses 4 chiral centres, however, to facilitate
synthetic ease the chirality about the hemiketal and the ester attached to the THP ring was not
conserved. The hemiketal group, although responsible for engaging in hydrogen bonding
with Glu208 (in both P. falciparum and human) was replaced with a methyl ketal to improve
stability. The free nitrogen atom was substituted with a paraꢀmethoxybenzyl (PMB) group,
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since benzyl substitution had been shown to improve the activity of latrunculin A;
inspection of the model suggested this group could be accommodated. The latrunculin core
analogue, compound 3b (Figure 1), preserves the thiazolidinone moiety of latrunculin with its
correct chirality.
Synthesis. Synthesis of the truncated latrunculin core analogue 3b, could potentially be
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achieved based on the synthesis of latrunculins by Fürstner et al.
or Smith et al. Here
we describe the successful synthesis of the latrunculin core analogues based on Smith et al.’s
total synthesis of (+)ꢀ18ꢀepiꢀlatrunculol A involving alkene metathesis as the key step and
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utilizing HoveydaꢀGrubbs’ II catalyst
(Figure 3). 3ꢀButenꢀ1ꢀol was TBS protected to
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obtain 5 and compound 7 was prepared according to literature in 5 steps. Compounds 5 and
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were then subjected to alkene metathesis and then treated with 6 M aqueous HCl to effect
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Michael addition of water to the α,βꢀunsaturated ketone, TBS deprotection and spontaneous
ring closure to obtain 9. The hemiketal 9 was then treated with catalytic camphor sulfonic
acid in methanol to obtain the methyl ketal 3b (the latrunculin core), which was esterified
using EDCI coupling to generate a focus set of analogues (Figure 4). It was found that similar
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to literature precedence, the orientation of the secondary alcohol in the methyl ketal 3b is
largely conserved thereby resulting in the isolation of two diastereomers of 3b (in 1:1 ratio)
instead of four.
Figure 3. Synthesis of the latrunculin core analogues based on the total synthesis of (+)ꢀ18ꢀ
epiꢀlatrunculol A.
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Figure 4. The focus set of modified latrunculin core analogues.
P. falciparum bloodꢀstage growth inhibition assay and identification of truncated
latrunculin analogues with low micromolar activity. The potency of the core analogues
against P. falciparum parasites was determined by monitoring their effect on the parasite life
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cycle using a growth inhibition assay.
Latrunculin B was found to be twice as potent as
the latrunculin core analogue 3b (Table 1), and the introduction of a butyl chain (10), a
cyclopentane derivative (11) or a furan ring (12) only led to slight improvement in the
activity. The benzoate derivative 13 was the first promising analogue synthesised in this
series, with an EC of 31 µM. The benzoate derivatives were explored further through
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Table 1. EC50 of actin inhibitors in P. falciparum bloodꢀstage replication.
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Compound
Latrunculin B
3b
EC50 (µM)
44 ± 2
>100
Compound
EC50 (µM)
7 ± 0.5
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*Data are the mean and standard deviation from a duplicate experiment.
The ortho iodo compound 14 was twice more potent than the meta and para iodo
compounds 19 and 21 (respectively) and thus the ortho position was further explored. The
higher potency of the ortho substituents was expected since it could mimic the presence of Z
double bond in the natural product. This led to the synthesis of analogues 15 ꢀ 17 with 6ꢀfold
increase in activity (7 µM). It was observed that the presence of a linear chain off the ortho
position 18 was less favoured than any of the closed ring systems.
With the meta substituted benzoate analogues, there was observed a twoꢀfold increase in
activity when the meta position had a thiophene ring (20) rather than an iodine atom (19).
Similar observations were also made at the para position implying that a bulkier hydrophobic
group was more favoured at these positions.
The activity was not greatly affected when methyl groups were incorporated at the ortho
position of the substituted benzoate analogues (23ꢀ25); this meant that although further
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substitution using small groups along the phenyl ring were tolerated in these compounds, it
did not improve the potency.
Invasion inhibition assay on Toxoplasma gondii (T.gondii) parasites. In the absence of
an assay to directly determine if the latrunculin core analogues were inhibitory by specifically
targeting P. falciparum actin in cultured bloodꢀstage parasites, we decided to test the
compounds against another parasite that shares a similar actinꢀbinding site. T.gondii is the
causative agent for toxoplasmosis, a widespread parasitic disease primarily affecting animals
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but which can also cause zoonotic infections in humans. Like P. falciparum, T. gondii
parasites are members of the Phylum Apicomplexa and share many of the conserved features
of actomyosinꢀdependent motility. As such, they are an attractive model for studying
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conserved features, such as actinꢀdependent motility and hostꢀcell invasion.
Analysis of
the structure of the ATP binding pocket of T.gondii and P. falciparum actins show they are
identical (Supplementary Information, Figure S2). A selection of the latrunculin core
analogues were then assayed in an inhibition of invasion assay to determine the potency of
these analogues toward blocking invasion of T. gondii parasites into host fibroblast cells
(Table 2).
Table 2. Selection of latrunculin core analogues and their corresponding activity against T.
gondii parasites.
*
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EC (µM)
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>100
>100
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6
19 ± 1
16 ± 1
1
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Data are the mean and standard deviation from a three individual experiments performed in triplicate.
As can be seen from Table 2, the latrunculin core analogues follow the same trend in
activity with the T. gondii as they did with the P. falciparum. The ester analogues with alkyl
chain (10) and cyclopentane ring (11) were inactive while the benzoate ester analogues with
ortho substitutions (15ꢀ17) were active. The fact that the activity in the P. falciparum is
mimicked by the T. gondii parasites supports the notion that these truncated latrunculin
analogues likely target the apicomplexan actin as their binding site.
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Determination of selectivity profile for the truncated latrunculin analogues. In order to
determine the selectivity profile of these analogues, pyrene fluorescence and cytotoxicity
assays were carried out using mammalian actin. Pyrene fluorescence is a powerful approach
used to study actin filament formation based on the enhancement of fluorescence when the
2
2, 35
pyreneꢀlabelled Gꢀactin (monomers) assemble to form the pyrenylated actin filaments.
Use of mammalian actin provides an indication of the selectivity of the truncated analogues
for Plasmodium parasites over mammalian actin (i.e. the degree of hostꢀeffect that might be
occurring in an invasion context). Table 3 represents the results of the pyrene fluorescence
22
using mammalian actin. It was observed that none of the truncated analogues demonstrated
a measurable decrease in the fluorescence (and thus polymerisation), indicating that these
compounds do not inhibit actin filament formation, and would therefore be expected to have
little effect on mammalian cells in culture. With the potent truncated analogues in the growth
inhibition assay being inactive in the pyrene fluorescence assay, and the fact that latrunculin
B shows 100% inhibition at a 10 µM concentration in the pyrene fluorescence assay (positive
control), these combined data suggest that these synthetic truncated analogues demonstrate
significant selectivity for the parasite over the mammalian actin.
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Table 3. A selection of analogues with their respective percentage inhibition of mammalian
actin polymerization.
Compound
Inhibition at 10 µM
Inhibition at 100 µM
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Latrunculin B
100%
<10%
<10%
<10%
<10%
100%
<10%
<10%
<10%
<10%
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Table 4 shows the results of cytotoxicity assay on a selection of truncated analogues. It can
be seen that the CC50 values of all these compounds were ~100 µM indicating that none of
these compounds showed any cytotoxicity with either the HEK293 or the HepG2 cell lines.
These results showed that the truncated latrunculins were very selective towards the P.
falciparum parasites over the mammalian and cancer cell lines.
Table 4. A selection of modified analogues with their respective cytotoxicity on HEK293 and
HepG2 cell lines.
Cytotoxicity CC (µM)
SI
50
Compound
10
P.falciparum P.falciparum
HEK293
HepG2
vs HEK293
vs HepG2
103
109
111
123
1
1
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6
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14
17
15
15
1
99
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SAR studies. A model of compound 15 in complex with P. falciparum actin (Figure 5) was
used to establish an understanding of possible binding interactions and SAR about the
truncated latrunculin analogues. As can be seen in Figure 5, the truncated latrunculin
derivative 15 successfully forms interactions with Tyr70 and Asn17 as observed in the model
of latrunculin B, however, interactions with Asp158 and Glu208 are lost due to PMB
protection of the nitrogen atom and the methoxylation of the stereogenic center, respectively,
as expected. Additionally, the hydrogen bond between the hydroxyl of Thr187 was also lost
in this model, replaced by a waterꢀmediated hydrogen bond with the carboxylate of the sideꢀ
chain of Glu188 (not shown). The phenylthiophene group replaces the m acrocycle of
latrunculin B, interacting across an extensive hydrophobic surface with Pro33 and Ile35.
Compound 15 achieves additional hydrophobic interactions from a π stacking interaction
between the sideꢀchain of Tyr70 and the PMB group. Thus, while protection of the ketal
oxygen and thiazolidinone nitrogen of the latrunculin core necessarily removed key
interactions, these were replaced by new interactions that likely account for the increased
activity of 15 over latrunculin B in P. falciparum parasites. Critically, the interaction with the
single residue difference between P. falciparum and human actin, Asn17, is retained,
accounting for the selectivity profile observed.
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Figure 5. Predicted binding interactions of the truncated latrunculin derivative 15 with P.
falciparum actin. Carbon atoms of compound 15 are coloured green, and the interacting actin
residues in tan; hydrogen atoms are white, nitrogen blue, and oxygen red. Hydrogen bonds
between ligand and receptor are represented as dashed lines.
The surface slice of the P. falciparum actin model with 15 bound (Figure 6) illustrates how
the ligand is predicted to be accommodated in the binding pocket. One edge of the
phenylthiophene is exposed to solvent, permitting substitution of this group with larger
moieties (eg. compounds 17, 23ꢀ25). Smaller groups at this position, such as compounds 3b,
10ꢀ12 present a significantly smaller hydrophobic surface and consequently reduced binding
affinity. The PMB packs against the hydrophobic surface offered by the sideꢀchain of Tyr70;
one edge of this group is solvent exposed, permitting exploration of larger functionalities at
this position.
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Figure 6. Excluded volume surface of the ligandꢀbinding site in the model of truncated
latrunculin derivative 15 bound to P. falciparum actin.
CONCLUSIONS
We have used homology modelling to elucidate the structure of latrunculin B bound to
human and P. falciparum actin and have identified key differences near the ATP binding
pocket. We report herein the steps towards the creation of actin inhibitors as developmental
drugs for use with malaria, revealing simplified yet relatively potent and selective versions of
the latrunculins that could be developed further into experimental antimalarials. Our SAR
studies have led to the development of compounds 15ꢀ17, each showing a 6ꢀfold increase in
activity against P. falciparum parasites and also an excellent selectivity profile against
mammalian cell lines. Computational modelling of compound 15 bound to actin explains and
validates the key interactions and SAR of these truncated latrunculin analogues.
Developments in crystallography with heterologously expressed parasite actin to explore
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compound binding in vitro or the use of CRISPR/Cas9 for targeted mutagenesis to in vivo
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binding, present possibilities for developing compounds like 15 further into realisable
antimalarials of the future.
EXPERIMENTAL SECTION
1
. Molecular Modelling. Homology modelling used the MODELLER program using the
structure of rabbit muscle actin with latrunculin B bound as the template. His73 in the
template is postꢀtranslationally modified (methylation); however, methylation of actin
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in P. falciparum is absent. Surface exposed water molecules surrounding the template
were removed, leaving only water molecules within the ligand binding region and those
buried within the protein. ATP and an associated calcium ion were also included in the
models.
The lowest energy homology models of the human and the P. falciparum actin were then
37
subjected to energy minimisation using the program YASARA. The simulation cell was
defined to extend 5 Å beyond all atoms, the cell boundaries were set to periodic and the cell
filled with water. The YAMBER3 force field was used and the pressure was maintained at 1
bar. The energyꢀminimised models were then subjected to pKa prediction and cell
neutralisation, followed by a very short molecular dynamics simulation of 200 ps. The
models were then subjected to energy minimisation to obtain the final structures. Figures
38
were created using the CHIMERA program.
There are 4 possible diastereomers for 15, with unresolved chiral centres about the
hemiketal and THP ester linkage. The model of 15 bound to P. falciparum actin assumed the
THP was in the chair conformation, with the phenylthiophene ester and thiazolidinone
substituents in the equatorial position.
2. P. falciparum growth inhibition assay. The parasites chosen for this assay were at the
mature pigmented trophozoite stage, and were adjusted to a starting parasitemia of
0
.1% and 2% hematocrit. To 45 mL of infected red blood cells, 5 mL of stock
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compound was added and mixed to generate a final culture volume of 50 mL. Parasites
30
were allowed to develop through two cycles of erythrocyte invasion for 72 h at 37°C.
Glutaraldehyde (ProSciTech) was then added to the early trophozoite stage parasites
after the second round of invasion, in order to fix them for 1 hour at room temperature
(final concentration of 0.25% (v/v)). After fixation, the parasites were washed in human
tonicity phosphateꢀbuffered saline (HTPBS), stained with 106 SYBR green dye
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(Invitrogen) and 56105 red blood cells counted per well using a BD FACSCantoII
flowꢀcytometer. Fluorescence Activated Cell Sorting (FACS) counts were analysed
using FlowJoTM(Ver 6.4.7) software (Treestar). All growth inhibition assays were run
in a 96ꢀwell plate format with each antibody tested in triplicate wells.
3.
Invasion inhibition assay of T. gondii. Inhibition of invasion of Toxoplasma parasite
growth was determined using the 2F clone of the type I RH strain that expresses the E.
coli βꢀgalactoside enzyme (βꢀGal). A 10 mM stock solution was prepared by dissolving
the compounds in DMSO and then diluted in medium containing 1% DMSO, which
also served as a (compound free) control. The positive control used was the pyrazolo
[
3,4ꢀd] pyrimidine inhibitor. The T. gondii parasites were then mixed with the
compounds and preincubated for 20 min at room temperature before being used to
challenge confluent monolayers of human foreskin fibroblasts (HFF) grown in 96ꢀwell
plates containing Dulbecco's Modified Eagle Medium (DMEM) supplemented with
1
0% Fetal bovine serum (FBS). All compounds were subjected to three individual
2
experiments, each performed in triplicates. Following addition of 5 × 10 parasites/well
containing dilutions of compounds and/or 1% DMSO, plates were centrifuged at
∼300g
for 5 min and returned to culture at 37°C, 5% CO . Data were analysed using Prism
2
(GraphPad) to determine EC50 values by plotting normalized, log transformed data (X
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axis), using nonlinear regression analysis as a sigmoidal doseꢀresponse curve with
variable slope.
4
. Pyrene Fluorescence assays. Unlabelled actin was mixed with pyreneꢀlabelled actin to
give a 5% labelled stock stored in calcium buffer G (CaBG: 2 mM Tris pH 8, 0.2 mM
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ATP, 0.5 mM DTT, 0.1 mM CaCl , 1 mM NaAzide). The stock was incubated for 2
2
min at room temperature with 10 × ME (final concentration 1 mM ethylene glycol
2
teraacetic acid (EGTA), 0.1 mM MgCl ) and 100 × AF (antifoam 204, Sigma) in order
2
+
to convert to Mg salt immediately prior to polymerization. This gave a final actin
concentration of 2.5 µM. The pyreneꢀlabelled actin monomer was incubated for a
further 1 min with 5 µl of each compound dissolved in DMSO or latrunculin B
(
positive control) to a final concentration of 1 µM, 10 µM or 100 µM. Polymerisation
was induced by addition and mixing of 10 × KMEI (final concentration 50 mM KCl, 10
mM imidazole pH 7, 1 mM MgCl , 1 mM EGTA), with the remaining volume made up
with magnesium buffer G (MgBG: as CaBG but 0.1 mM MgCl in place of CaCl ).
2
2
2
Immediately after, pyrene fluorescence (excitation 365 nm, emission 407 nm) was
monitored in a spectrofluorometer (Tecan Infinite M200 Pro) every 15 s for 90 min.
Each assay was run in triplicate.
5
. Cytotoxicity Assay. HEK293 (human embryonic kidney) and HepG2 (Hepatocellular
carcinoma) cells were seeded as 3000 and 5000 cells per well, respectively, in a clear
3
#
5
84ꢀwell plate in a final volume of 20 µl in DMEM medium (GIBCOꢀInvitrogen
11995ꢀ073), in which 10% of FBS was added. Cells were incubated for 24 h at 37°C,
2
% CO to allow cells to attach to the plates. Compounds were prepared at 20 mM in
100% DMSO. The highest concentration to test the compounds was 100 µM, in order
to maintain the DMSO final concentration at 0.5%. A dilution series 1:3 fold steps in
serum free medium was created, and 20µl of each dilution was added to the cell culture
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wells, resulting in 5% FBS as final concentration. 100 µM tamoxifen served as negative
control (all cells die) and in addition a serial dilution of tamoxifen, started at 100 µM,
was included as a dose response control. The cells were incubated with the compounds
for 24 h at 37°C, 5% CO . After the incubation, 10 µM resazurin (dissolved in
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Phosphate Buffered Saline (PBS)) was added to each well. The plates were then
2
incubated for 3 h at 37°C, 5% CO . The fluorescence intensity (FI) was read using
Polarstar Omega plate reader with excitation/emission 560/590. The data was analysed
by Prism software. Results are presented as the average percentage of control for each
set of triplicate wells using the following equation: Percentage Viability = ((FITEST
–
FINegative)/(FIUNTREATED – FINegative))*100. The selectivity index was calculated using SI
=
CC50/EC50.
6
. General Chemistry Experimental. Solvents were of analytical grade: ethyl acetate
(EtOAc); dichloromethane (DCM); dimethyl formamide (DMF); methanol (MeOH);
tetrahydrofuran (THF). Analytical TLC was performed on silica gel 60/F254 preꢀcoated
aluminium sheets (0.25 mm, Merck). Flash column chromatography was carried out
1
13
with silica gel 60, 0.63–0.20 mm (70–230 mesh, Merck). H and C NMR spectra were
recorded at 400.13, and 100.62 MHz, respectively, on a Bruker Avance III Nanobay
spectrometer with BACS 60 sample changer, using solvents from Cambridge Isotope
Laboratories. Chemical shifts (δ, ppm) are reported relative to the solvent peak (CDCl3:
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.26 [ H] or 77.16 [ C], DMSOꢀd : 2.50 [ H] or 39.52 [ C]). Proton resonances are
6
annotated as: chemical shift (ppm), multiplicity (s, singlet; d, doublet; t, triplet; q,
1
quartet; m, multiplet), coupling constant (J, Hz), and number of protons. The H NMR
spectra of diastereomer mixtures have been described separately for both diastereomers
where distinct signals could be assigned. In cases where the peaks of both the
diastereomers overlapped, they have been described as a single NMR spectrum.
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Analytical HPLC was acquired on an Agilent 1260 Infinity analytical HPLC coupled
with a G1322A degasser, G1312B binary pump, G1367E high performance
autosampler, G4212B diode array detector. Conditions: Zorbax Eclipse Plus C18 Rapid
resolution column (4.6 × 100 mm) with UV detection at 254 nm and 214 nm, 30°C;
sample was eluted using a gradient of 5 – 100% of solvent B in solvent A where solvent
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A: 0.1% aq. TFA, and solvent B: 0.1% TFA in CH
3
CN (5 to 100% B [9 min], 100% B
[
1 min]; 0.5 ml/min). Low resolution mass spectrometry was performed on an Agilent
6
100 Series Single Quad LCMS coupled with an Agilent 1200 Series HPLC, G1311A
quaternary pump, G1329A thermostatted autosampler, and G1314B variable
wavelength detector (214 and 254 nm). LC conditions: Phenomenex Luna C8(2)
column (100 Å, 5 µm, 50 × 4.6 mm), 30°C; sample (5 µL) was eluted using a binary
2 2 3
gradient (solvent A: 0.1% aq. HCO H; solvent B: 0.1% HCO H in CH CN; 5 to 100%
B [10 min], 100% B [10 min]; 0.5 ml/min). MS conditions: quadrupole ion source with
multimodeꢀESI; drying gas temperature, 300°C; vaporizer temperature, 200°C;
capillary voltage, 2000 V (positive mode) or 4000 V (negative mode); scan range, 100–
1
000 m/z; step size, 0.1 s over 10 min. High resolution MS was performed on an
Agilent 6224 TOF LCMS coupled to an Agilent 1290 Infinity LC. All data were
acquired and reference mass corrected via a dualꢀspray electrospray ionisation (ESI)
source. Each scan or data point on the total ion chromatogram (TIC) is an average of
1
3,700 transients, producing a spectrum every second. Mass spectra were created by
averaging the scans across each peak and subtracting the background from first 10 s of
the TIC. Acquisition was performed using the Agilent Mass Hunter Data Acquisition
software ver. B.05.00 Build 5.0.5042.2 and analysis was performed using Mass Hunter
Qualitative Analysis ver. B.05.00 Build 5.0.519.13. Acquisition parameters: mode, ESI;
drying gas flow, 11 L/min; nebuliser pressure, 45 psi; drying gas temperature, 325°C;
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voltages: capillary, 4000 V; fragmentor, 160 V; skimmer, 65 V; octapole RF, 750 V;
scan range, 100–1500 m/z; positive ion mode internal reference ions, m/z 121.050873
and 922.009798. LC conditions: Agilent Zorbax SBꢀC18 Rapid Resolution HT (2.1 ×
5
0 mm, 1.8 µm column), 30°C; sample (5 µL) was eluted using a binary gradient
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(solvent A: 0.1% aq. HCO H; solvent B: 0.1% HCO H in CH CN; 5 to 100% B [3.5
2
2
3
1
min], 0.5 ml/min). Purity was determined either by analytical HPLC or by H NMR and
was found to be ≥95% for all compounds.
General Procedure A: EDCI coupling. To the alcohol (1 eq.) dissolved in DCM (0.02
M), was added the carboxylic acid (2.8 eq.), DMAP (3 eq.) and EDCI.HCl (3 eq.). The
solution was then stirred at room temperature till the completion of the reaction, as indicated
by the TLC analysis. Saturated ammonium chloride solution was then added and the aqueous
layer extracted with DCM (x 3). The combined organic layer was then dried with sodium
sulfate and evaporated to dryness. Flash chromatography, eluting with 15% EtOAc/petroleum
spirits yielded the product.
General procedure B: Suzuki coupling. To the corresponding halide (1 eq.) dissolved in
4
:1 ratio of 1,4ꢀdioxane and water (0.05 M), was added potassium carbonate (3 eq.). The
solution was degassed to remove all the dissolved oxygen from the solution, before catalytic
amount of Pd(dppf)Cl (0.05 eq.) was added and the reaction mixture refluxed for 12 h. The
2
reaction was allowed to cool to room temperature and then filtered through celite to remove
most of the catalyst. The filtrate was then diluted with EtOAc and washed with brine. The
organic layer was dried with sodium sulfate and evaporated to dryness. Flash
chromatography, eluting with 15% EtOAc/petroleum spirits yielded the desired product.
4
ꢀ(4ꢀHydroxyꢀ2ꢀmethoxytetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)ꢀ3ꢀ(4ꢀmethoxybenzyl)thiazolidinꢀ
2
ꢀone (3b)
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4ꢀ(2,4ꢀdihydroxytetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)ꢀ3ꢀ(4ꢀmethoxybenzyl)thiazolidinꢀ2ꢀone 9 (1.14
g, 3.36 mmol) was dissolved in the corresponding alcohol (0.05 M) and catalytic CSA (0.1
eq.) was added. The reaction was stirred for 12 h before being quenched with sodium
bicarbonate solution and extracted repeatedly with DCM. The combined organic layer was
washed with brine and dried with sodium sulfate before evaporating to dryness to yield the
product as clear oil as a mixture of two diastereomers; A and B in 1:1 ratio respectively (826
mg, 69%).
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59
60
HPLC – rt 5.96 min > 98% purity at 254 nm; LRMS (only mass ion for the fragment
+
obtained after elimination of water and methanol was observed) [Mꢀ(H O + MeOH)] 304.1
2
+
1
m/z; HRMS [M+H] 354.137 m/z, found 354.1355 m/z; H NMR (diastereomer A) (400
MHz, CDCl3) δ 7.23 – 7.22 (m, 2H), 6.91 – 6.82 (m, 2H), 5.20 (d, J = 15.4 Hz, 1H), δ 4.28
(d, J = 14.4 Hz, 1H), 4.11 – 3.99 (m, 1H), 3.97 – 3.91 (m, 1H), 3.86 – 3.81 (m, 1H), 3.79 (d, J
=
1.7 Hz, 3H), 3.66 – 3.57 (m, 1H), 3.39 – 3.30 (m, 1H), 3.29 – 3.18 (m, 1H), 3.08 (s, 3H),
2.20 (ddd, J = 12.6, 4.6, 1.8 Hz, 1H), 1.98 – 1.88 (m, 1H), 1.67 – 1.66 (m, 1H), 1.54 – 1.38
1
(
m, 1H); H NMR (diastereomer B) (400 MHz, CDCl3) 7.14 – 7.12 (m, J = 8.5 Hz, 2H), 6.91
– 6.82 (m, 2H), 5.02 (d, J = 14.3 Hz, 1H), 4.20 (d, J = 15.5 Hz, 1H), 4.11 – 3.99 (m, 1H),
3.88 – 3.87 (m, 1H), 3.86 – 3.81 (m, 1H), 3.79 (d, J = 1.7 Hz, 3H), 3.57 – 3.48 (m, 1H), 3.39
– 3.30 (m, 1H), 3.29 – 3.18 (m, 1H), 3.01 (s, 3H), 2.10 (ddd, J = 12.8, 4.7, 1.8 Hz, 1H), 1.98
13
– 1.88 (m, 1H), 1.55 – 1.48 (m, 1H), 1.54 – 1.38 (m, 1H); C NMR (101 MHz, CDCl ) δ
3
1
59.2, 130.2, 129.0, 128.6, 114.3, 114.1, 103.3, 64.5, 60.5, 60.3, 59.3, 56.9, 55.4, 47.8, 47.6,
7.5, 46.8, 38.0, 37.35, 34.8, 34.5, 26.2, 25.4.
4
3
9
(Butꢀ3ꢀenꢀ1ꢀyloxy)(tertꢀbutyl)dimethylsilane (5)
To 3ꢀbutenꢀ1ꢀol 4 (3ml, 34.9 mmol) in DCM (90 ml), imidazole (4.8 mg, 70 mmol) and
TBSCl (5.8 g, 38.3 mmol) was added and stirred for 20 h. On completion of the reaction,
water was added to quench the reaction followed by extraction with DCM (x 3). The
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combined organic layer was washed with ice cold 1 M aq. HCl and then with water (x 3). The
organic layer was then dried with sodium sulfate, filtered and evaporated to dryness to yield
the product (6.48 g, 99% yield).
1
H NMR (400 MHz, CDCl ) δ 5.82 (ddt, J = 17.1, 10.2, 6.9 Hz, 1H), 5.16 – 4.95 (m, 2H),
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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9
0
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8
9
0
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8
9
0
3.66 (t, J = 6.8 Hz, 2H), 2.27 (qt, J = 6.8, 1.3 Hz, 2H), 0.89 (s, 9H), 0.05 (s, 6H).
25
4ꢀAcryloylꢀ3ꢀ(4ꢀmethoxybenzyl)thiazolidinꢀ2ꢀone (7)
To Nꢀmethoxyꢀ3ꢀ(4ꢀmethoxybenzyl)ꢀNꢀmethylꢀ2ꢀoxothiazolidineꢀ4ꢀcarboxamide 6 (641
mg, 2.06 mmol) in THF (7ml) at 0°C, 1.5 M vinylmagnesium bromide (7.5 mL, 10.3 mmol)
was added drop wise and stirred overnight. After completion of the reaction, 2 M aq. HCl
was added and then the aqueous layer extracted with DCM (x 3). The combined organic layer
was washed with saturated sodium bicarbonate solution, dried with sodium sulfate and
evaporated to dryness. Flash chromatography, eluting with a gradient from 10% to 50%
EtOAc/petroleum spirits led to the isolation of the product (436 mg, 93%).
1
H NMR (400 MHz, CDCl ) δ 7.17 – 7.03 (m, 2H), 6.89 – 6.77 (m, 2H), 6.48 (dd, J = 17.4,
3
1
0.4 Hz, 1H), 6.35 (dd, J = 17.4, 1.3 Hz, 1H), 5.90 (dd, J = 10.4, 1.3 Hz, 1H), 5.06 (d, J =
4.7 Hz, 1H), 4.33 (dd, J = 9.4, 4.5 Hz, 1H), 3.82 (d, J = 14.8 Hz, 1H), 3.79 (s, 3H), 3.50 (dd,
1
J = 11.5, 9.4 Hz, 1H), 3.13 (dd, J = 11.5, 4.5 Hz, 1H).
)ꢀ4ꢀ(5ꢀ((tertꢀButyldimethylsilyl)oxy)pentꢀ2ꢀenoyl)ꢀ3ꢀ(4ꢀmethoxybenzyl)thiazolidinꢀ2ꢀ
one (8)
To 4ꢀacryloylꢀ3ꢀ(4ꢀmethoxybenzyl)thiazolidinꢀ2ꢀone 7 (661 mg, 2.9 mmol) dissolved in
(E
DCE (16 mL) was added (butꢀ3ꢀenꢀ1ꢀyloxy)(tertꢀbutyl)dimethylsilane 5 (1.1 g, 5.8 mmol)
and catalytic HoveydaꢀGrubbs’ II catalyst (91 mg, 0.145 mmol). The reaction was then
heated to 50°C for 12 h. Upon completion of the reaction, as indicated by TLC analysis, the
reaction mixture was filtered through celite to remove the catalyst and then subjected to flash
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chromatography, eluting with 10% EtOAc/petroleum spirits to obtain the product (693 mg,
53%).
+
HPLC – rt 4.03 min > 99% purity at 254 nm; LRMS [M+H] no mass ion was detected;
+
1
HRMS [M+H] 436.1972 m/z, found 436.1980 m/z; H NMR (400 MHz, CDCl ) δ 7.10 (d, J
3
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11
12
13
14
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= 8.6 Hz, 2H), 7.00 (dt, J = 15.7, 7.0 Hz, 1H), 6.83 (d, J = 8.7 Hz, 2H), 6.25 (dt, J = 15.7, 1.4
Hz, 1H), 5.05 (d, J = 14.8 Hz, 1H), 4.27 (dd, J = 9.3, 4.6 Hz, 1H), 3.81 (d, 1H), 3.78 (s, 3H),
3
–
1
.72 (t, J = 6.1 Hz, 2H), 3.46 (dd, J = 11.4, 9.4 Hz, 1H), 3.11 (dd, J = 11.4, 4.6 Hz, 1H), 2.48
13
2.34 (m, J = 6.3, 1.4 Hz, 2H), 0.87 (s, 9H), 0.04 (s, 6H); C NMR (101 MHz, CDCl
3
) δ
94.8, 172.1, 159.5, 148.7, 130.1, 127.5, 126.3, 114.3, 63.9, 61.2, 55.4, 47.3, 36.3, 28.0, 25.9,
18.3, ꢀ5.3.
ꢀ(2,4ꢀDihydroxytetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)ꢀ3ꢀ(4ꢀmethoxybenzyl)thiazolidinꢀ2ꢀone (9)
(E)ꢀ4ꢀ(5ꢀ((tertꢀbutyldimethylsilyl)oxy)pentꢀ2ꢀenoyl)ꢀ3ꢀ(4ꢀmethoxybenzyl)thiazolidinꢀ2ꢀ
4
(
one)ꢀ4ꢀ(5ꢀ((tertꢀbutyldimethylsilyl)oxy)pentꢀ2ꢀenoyl)ꢀ3ꢀ(4ꢀmethoxybenzyl)thiazolidinꢀ2ꢀone
8 (1.72 mg, 3.95 mmol) was dissolved in THF (0.05 M) and 1 M aq. HCl (0.35 M) and stirred
for 12 h. The reaction was then quenched with sodium bicarbonate solution and the aqueous
layer washed with DCM. The combined organic layer was washed with brine, dried with
sodium sulfate and evaporated in vacuo. Flash chromatography eluting with 100% diethyl
ether yielded the desired product as clear oil and was immediately used for the next step.
(1.14 g, 85%).
2ꢀMethoxyꢀ2ꢀ((
R)ꢀ3ꢀ(4ꢀmethoxybenzyl)ꢀ2ꢀoxothiazolidinꢀ4ꢀyl)tetrahydroꢀ2Hꢀpyranꢀ4ꢀ
yl pentanoate (10)
Title compound was prepared from 4ꢀ(4ꢀhydroxyꢀ2ꢀmethoxytetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)ꢀ3ꢀ
(4ꢀmethoxybenzyl)thiazolidinꢀ2ꢀone 3b (40 mg, 0.113 mmol) and valeric acid (37 µL, 0.34
mmol) according to the General Procedure A as a clear oil and a mixture of two
diastereomers; A and B in 1:0.6 ratio respectively (40.4 mg, 82%).
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+
+
HPLC – rt 8.62 min > 99% purity at 254 nm; LRMS [M+Na] 460.2 m/z; HRMS [M+H]
1
4
38.1945 m/z, found 438.1950 m/z; H NMR (diastereomers A) (400 MHz, CDCl
3
) δ 7.25 –
7
.19 (m, 2H), 6.90 – 6.82 (m, 2H), 5.14 – 5.13 (m, 1H), 5.05 (d, J = 14.4 Hz, 1H), 4.28 (d, J
14.4 Hz, 1H), 3.99 – 3.86 (m, 1H), 3.84 – 3.83 (m, 1H), 3.80 (s, 3H), 3.68 – 3.67 (m, 1H),
3.37 – 3.36 (m, 1H), 3.25 – 3.24 (m, 1H), 3.10 (s, 3H), 2.34 – 2.25 (m, 2H), 2.19 (ddd, J =
=
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0
6
–
2.5, 4.8, 1.9 Hz, 1H), 1.98 – 1.97 (m, 1H), 1.69 – 1.46 (m, 4H), 1.35 – 1.34 (m, 2H), 0.96 –
1
.88 (m, 3H); H NMR (diastereomers B) (400 MHz, CDCl
3
) δ 7.14 – 7.13 (m, 2H), 6.90 –
.82 (m, 2H), 5.21 (d, J = 15.4 Hz, 1H), 5.14 – 5.13 (m, 1H), 4.20 (d, J = 15.5 Hz, 1H), 3.99
3.86 (m, 1H), 3.84 – 3.83 (m, 1H), 3.80 (s, 3H), 3.64 – 3.55 (m, 1H), 3.25 (m, 2H), 3.02 (s,
3H), 2.34 – 2.25 (m, 2H), 2.09 (ddd, J = 12.8, 4.9, 1.9 Hz, 1H), 1.98 – 1.97 (m, 1H), 1.80 (dd,
13
J = 12.7, 11.3 Hz, 1H), 1.69 – 1.46 (m, 3H), 1.35 – 1.34 (m, 2H), 0.96 – 0.88 (m, 3H); C
NMR (101 MHz, CDCl ) δ 173.4, 173.0, 159.2, 159.2, 130.2, 128.9, 128.6, 128.6, 114.3,
14.1, 103.2, 102.4, 67.0, 67.0, 60.1, 59.9, 59.2, 56.7, 55.4, 55.4, 47.7, 47.6, 47.5, 46.8, 34.4,
34.4, 34.3, 33.7, 31.2, 27.2, 26.2, 25.3, 22.4, 22.4, 13.9.
ꢀMethoxyꢀ2ꢀ(( )ꢀ3ꢀ(4ꢀmethoxybenzyl)ꢀ2ꢀoxothiazolidinꢀ4ꢀyl)tetrahydroꢀ2Hꢀpyranꢀ4ꢀ
3
1
2
R
yl cyclopentanecarboxylate (11)
Title compound was prepared from 4ꢀ(4ꢀhydroxyꢀ2ꢀmethoxytetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)ꢀ3ꢀ
(
4ꢀmethoxybenzyl)thiazolidinꢀ2ꢀone 3b (41 mg, 0.116 mmol) and cyclopentane carboxylic
acid (38 µL, 0.35 mmol) according to the General Procedure A as a clear oil and a mixture of
two diastereomers; A and B in 1:0.74 ratio respectively (20 mg, 38%).
HPLC – rt (diastereomer A) 9.268 min (55%), (diastereomer B) 9.297 min (45%),
+
collectively > 99% purity at 254 nm; LRMS no mass ion was detected; HRMS [M+H]
1
450.1945 m/z, found 450.1950 m/z; H NMR (diastereomers A) (400 MHz, CDCl
3
) δ 7.25 –
7
.19 (m, 2H), 6.89 – 6.82 (m, 2H), 5.17 – 5.07 (m, 1H), 5.04 (d, J = 14.4 Hz, 1H), 4.28 (d, J
=
14.4 Hz, 1H), 3.91 – 3.81 (m, 2H), 3.80 (s, 3H), 3.68 – 3.67 (m, 1H), 3.36 ꢀ 3.24 (m, 2H),
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3.10 (s, 3H), 2.76 – 2.61 (m, 1H), 2.21 – 2.13 (m, 1H), 1.97 – 1.96 (m, 1H), 1.92 –1.44 (m,
1
1
0H); H NMR (diastereomers B) (400 MHz, CDCl
3
) δ 7.13 – 7.12 (m, 2H), 6.89 – 6.82 (m,
2H), 5.21 (d, J = 15.5 Hz, 1H), 5.17 – 5.07 (m, 1H), 4.20 (d, J = 15.5 Hz, 1H), 3.95 – 3.94
(m, 1H), 3.91 – 3.81 (m, 1H), 3.79 (s, 3H), 3.63 – 3.54 (m, 1H), 3.36 ꢀ 3.24 (m, 2H), 3.02 (s,
10
11
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13
14
15
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59
60
3H), 2.76 – 2.61 (m, 1H), 2.07 (ddd, J = 12.8, 4.8, 1.8 Hz, 1H), 1.97 – 1.96 (m, 1H), 1.92 –
1
3
1
1
4
3
.44 (m, 10H); C NMR (101 MHz, CDCl ) δ 176.3, 176.2, 173.4, 173.1, 159.2, 159.2,
30.2, 129.0, 128.6, 128.6, 114.3, 114.1, 103.3, 102.5, 66.9, 66.9, 60.1, 60.0, 59.3, 56.8, 55.4,
7.7, 47.6, 47.5, 46.8, 44.0, 34.2, 33.6, 31.2, 30.2, 30.1, 30.1, 26.2, 26.0, 25.3.
2ꢀMethoxyꢀ2ꢀ((R)ꢀ3ꢀ(4ꢀmethoxybenzyl)ꢀ2ꢀoxothiazolidinꢀ4ꢀyl)tetrahydroꢀ2Hꢀpyranꢀ4ꢀ
yl furanꢀ2ꢀcarboxylate (12)
Title compound was prepared from 4ꢀ(4ꢀhydroxyꢀ2ꢀmethoxytetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)ꢀ3ꢀ
(4ꢀmethoxybenzyl)thiazolidinꢀ2ꢀone 3b (40 mg, 0.113 mmol) and 2ꢀfuroic acid (38 mg, 0.34
mmol) according to the General Procedure A as a white semisolid and a mixture of two
diastereomers; A and B in 1:0.65 ratio respectively (50.2 mg, 98%).
HPLC – rt 7.80 min > 95% purity at 254 nm; LRMS no mass ion was detected; HRMS
+
1
[
M+H] 448.1424 m/z, found 448.1424 m/z; H NMR (diastereomer A) (400 MHz, CDCl
3
) δ
.58 – 7.57 (m, 1H), 7.23 – 7.22 (m, 2H), 7.20 – 7.16 (m, 1H), 6.90 – 6.82 (m, 2H), 6.51 –
.50 (m, 1H), 5.37 – 5.36 (m, 1H), 5.07 (d, J = 14.4 Hz, 1H), 4.29 (d, J = 14.4 Hz, 1H), 4.01
7
6
– 3.89 (m, 1H), 3.89 – 3.81 (m, 1H), 3.79 (s, 3H), 3.77 – 3.69 (m, 1H), 3.42 – 3.35 (m, 1H),
3.32 – 3.18 (m, 1H), 3.12 (s, 3H), 2.30 (ddd, J = 12.5, 4.8, 1.9 Hz, 1H), 2.10 – 2.09 (m, 1H),
1
1
.80 (dd, J = 12.4, 11.5 Hz, 1H), 1.76 – 1.60 (m, 1H); H NMR (diastereomer B) (400 MHz,
CDCl
6.51 – 6.50 (m, 1H), 5.37 – 5.36 (m, 1H), 5.21 (d, J = 15.5 Hz, 1H), 4.20 (d, J = 15.5 Hz,
H), 4.01 – 3.89 (m, 1H), 3.89 – 3.81 (m, 1H), 3.80 (s, 3H), 3.69 – 3.58 (m, 1H), 3.32 – 3.18
3
) δ 7.58 – 7.57 (m, 1H), 7.20 – 7.16 (m, 1H), 7.17 – 7.11 (m, 2H), 6.90 – 6.82 (m, 2H),
1
(m, 2H), 3.04 (s, 3H), 2.20 (ddd, J = 12.7, 4.9, 1.9 Hz, 1H), 2.10 – 2.09 (m, 1H), 1.94 (dd, J =
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3
12.7, 11.3 Hz, 1H), 1.76 – 1.60 (m, 1H); C NMR (101 MHz, CDCl
3
) δ 173.3, 159.3, 158.2,
46.6, 144.7, 130.2, 128.9, 128.6, 118.4, 118.3, 114.3, 114.1, 112.0, 103.3, 102.5, 68.1, 68.0,
0.1, 59.9, 59.2, 56.8, 55.4, 47.8, 47.7, 47.5, 46.8, 34.3, 33.7, 31.3, 26.2, 25.3.
ꢀMethoxyꢀ2ꢀ(3ꢀ(4ꢀmethoxybenzyl)ꢀ2ꢀoxothiazolidinꢀ4ꢀyl)tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl
1
6
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
benzoate (13)
Title compound was prepared from 4ꢀ(4ꢀhydroxyꢀ2ꢀmethoxytetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)ꢀ3ꢀ
(
4ꢀmethoxybenzyl)thiazolidinꢀ2ꢀone 3b (52.7 mg, 0.15 mmol) and benzoic acid (55 mg, 0.45
mmol) according to the General Procedure A as a white semisolid as a mixture of two
diastereomers; A and B in 1: 0.8 ratio respectively (41 mg, 59%).
HPLC – rt 8.3 min > 99% purity at 254 nm; LRMS no mass ion was detected; HRMS
+
1
[
M+H] 458.1632 m/z, found 458.1646 m/z; H NMR (diastereomer A) (400 MHz, CDCl
3
) δ
.04 – 8.03 (m, 2H), 7.57 (t, J = 7.4 Hz, 1H), 7.45 (t, J = 7.7 Hz, 2H), 7.25 – 7.24 (m, 2H),
.91 – 6.83 (m, 2H), 5.47 – 5.32 (m, 1H), 5.08 (d, J = 14.4 Hz, 1H), 4.32 (d, J = 14.4 Hz,
8
6
1H), 3.90 – 3.82 (m, 2H), 3.81 (s, 3H), 3.79 – 3.72 (m, 1H), 3.43 – 3.38 (m, 1H), 3.32 – 3.24
(
(
(
(
m, 1H), 3.14 (s, 3H), 2.33 (ddd, J = 12.4, 4.7, 1.8 Hz, 1H), 2.17 – 2.08 (m, 1H), 1.87 – 1.77
1
m, 1H), 1.69 – 1.61 (m, 1H); H NMR (diastereomer B) (400 MHz, CDCl
3
) δ 8.04 – 8.03
m, 2H), 7.57 (t, J = 7.4 Hz, 1H), 7.45 (t, J = 7.7 Hz, 2H), 7.15 – 7.14 (m, 2H), 6.94 – 6.80
m, 2H), 5.49 – 5.32 (m, 1H), 5.23 (d, J = 15.5 Hz, 1H), 4.22 (d, J = 15.5 Hz, 1H), 3.97 –
3.96 (m, 2H), 3.80 (s, 3H), 3.72 – 3.62 (m, 1H), 3.33 – 3.21 (m, 2H), 3.06 (s, 3H), 2.24 (ddd,
J = 12.7, 4.8, 1.7 Hz, 1H), 2.16 – 2.07 (m, 1H), 1.96 (dd, J = 12.6, 11.3 Hz, 1H), 1.74 – 1.73
13
(
m, 1H); C NMR (101 MHz, CDCl
3
) δ 173.3, 173.1, 166.0, 166.0, 159.3, 159.2, 133.2,
1
30.2, 129.8, 129.8, 128.9, 128.6, 128.6, 128.5, 114.3, 114.1, 103.3, 102.5, 68.0, 67.8, 60.1,
59.9, 59.3, 56.8, 55.4, 47.8, 47.7, 47.6, 46.8, 34.3, 33.7, 31.3, 27.0, 26.2, 25.3.
ꢀMethoxyꢀ2ꢀ(3ꢀ(4ꢀmethoxybenzyl)ꢀ2ꢀoxothiazolidinꢀ4ꢀyl)tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl 2ꢀ
iodobenzoate (14)
2
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Page 29 of 49
Journal of Medicinal Chemistry
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Title compound was prepared from 4ꢀ(4ꢀhydroxyꢀ2ꢀmethoxytetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)ꢀ3ꢀ
(
4ꢀmethoxybenzyl)thiazolidinꢀ2ꢀone 3b (151 mg, 0.43 mmol) and 2ꢀiodobenzoic acid (318
mg, 1.28 mmol) according to the General Procedure A as a white semisolid and a mixture of
two diastereomers; A and B in 1:0.9 ratio respectively (151 mg, 60%).
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LRMS no mass ion was detected; HRMS [M+H] 584.0598 m/z, found 584.0594 m/z; H
NMR (diastereomer A) (400 MHz, CDCl ) δ 7.99 (d, J = 8.0 Hz, 1H), 7.79 (ddd, J = 7.8, 3.7,
.7 Hz, 1H), 7.42 (td, J = 7.5, 0.9 Hz, 1H), 7.24 – 7.23 (m, 1H), 7.19 – 7.12 (m, 2H), 6.87
dd, J = 8.8, 2.8 Hz, 2H), 5.49 – 5.38 (m, 1H), 5.05 (d, J = 14.3 Hz, 1H), 4.32 (d, J = 14.4 Hz,
H), 4.00–3.92 (m, 1H), 3.91 – 3.82 (m, 1H), 3.81 (s, 3H), 3.79 – 3.71 (m, 1H), 3.44 – 3.37
3
1
(
1
(m, 1H), 3.28 – 3.27 (m, 1H), 3.14 (s, 3H), 2.38 (ddd, J = 12.4, 4.8, 1.9 Hz, 1H), 2.23 – 2.11
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(
m, 1H), 1.87 – 1.79 (dd, J = 12.3, 11.5 Hz, 1H), 1.71 – 1.64 (m, 1H). H NMR (diastereomer
B) (400 MHz, CDCl
td, J = 7.5, 0.9 Hz, 1H), 7.26 – 7.21 (m, 1H), 7.19 – 7.11 (m, 2H), 6.87 – 6.86 (m, J = 8.8,
2.8 Hz, 2H), 5.52 – 5.34 (m, 1H), 5.23 (d, J = 15.5 Hz, 1H), 4.23 (d, J = 15.5 Hz, 1H), 4.03 –
3
) δ 7.99 (d, J = 8.0 Hz, 1H), 7.79 (ddd, J = 7.8, 3.7, 1.7 Hz, 1H), 7.42
(
3
3
1
1
.90 (m, 1H), 3.91 – 3.82 (m, 1H), 3.80 (s, 3H), 3.71 – 3.62 (m, 1H), 3.28 – 3.27 (m, 2H),
.06 (s, 3H), 2.27 (ddd, J = 12.8, 5.1, 1.9 Hz, 1H), 2.17 – 2.16 (m, 1H), 1.99 (dd, J = 12.7,
1
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1.3 Hz, 1H), 1.79 – 1.71 (m, 1H); > 95% purity by H NMR; C NMR (101 MHz, CDCl
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) δ
73.2, 172.9, 165.9, 165.8, 159.1, 159.1, 141.3, 141.3, 135.1, 134.9, 132.7, 132.7, 131.0,
130.9, 130.1, 130.1, 128.8, 128.5, 128.5, 128.0, 114.2, 114.0, 103.1, 102.4, 94.1, 94.0, 69.0,
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8.9, 59.9, 59.8, 59.3, 56.7, 55.3, 55.3, 47.7, 47.6, 47.6, 46.7, 34.1, 33.6, 31.1, 31.0, 26.1,
5.3.
2
2
ꢀMethoxyꢀ2ꢀ((R)ꢀ3ꢀ(4ꢀmethoxybenzyl)ꢀ2ꢀoxothiazolidinꢀ4ꢀyl)tetrahydroꢀ2Hꢀpyranꢀ4ꢀ
yl 2ꢀ(thiophenꢀ2ꢀyl)benzoate (15)
Title compound was prepared from 2ꢀmethoxyꢀ2ꢀ(3ꢀ(4ꢀmethoxybenzyl)ꢀ2ꢀoxothiazolidinꢀ
4
ꢀyl)tetrahydroꢀ2Hꢀpyranꢀ4ꢀyl 2ꢀiodobenzoate 14 (43.8 mg, 0.075 mmol) and thiopheneꢀ2ꢀ
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